Case Report: Decrypting an interchromosomal insertion associated with Marfan’s syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient’s neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.


Tables
Patient's score for Marfan syndrome according to Ghent criteria (*https://www.ncbi.nlm.nih.gov/books/NBK537339/ ) Systemic characteristics according to Ghent criteria* Score Wrist and thumb sign 3 Pectus excavatum or chest asymmetry 1 Reduced upper segment/lower segment ratio and increased arm span/height and no severe scoliosis 1

Scoliosis or thoracolumbar kyphosis 1
Reduced elbow extension (equal to 170 degrees with full extension) 1

Mitral valve prolapse 1
Total score 8    Figure S3: Fusion transcript PARP4-RNF17 at sequence breakpoint joining fragments 13B(+) + 13C(-) (see Bjct2 in Table 1)   S3).Discordant reads that map to the left (A, B) or right (C, D) region of the breakpoint have mate pairs that map to the positive (+) or negative (-) strand at a second chromosome region.Genomic coordinates of soft-clipped reads are highlighted within boxes.Breakpoint junction sequences, of which two characterized by microhomology of 3-5 bps (A, C) and one by blunt ends (B), are indicated.
Figure S5 IGV exploration of the fragments 4B and 13D that compose the derivative chromosome 4. Dashed vertical lines indicate the breakpoints of each fragment as identified by OGM.Soft-clipped reads that map to the right side of the breakpoint at chr4:44104800 (+) have mate pair located to negative (-) strand of chromosome 13 (fragment 13D inv).Blast results of soft-clipped read sequences at chromosome 13 were not informative due to the presence of repeated sequences.
Figure S6 IGV visualization of fragments 3Ainv and 13A (indicated with rectangles) that compose the derivative chromosome 13.Dashed vertical lines indicate the breakpoints of each fragment as identified by OGM.Soft-clipped reads that map to the left side of the breakpoint at chr13:23529137(+) have mate pair located to negative (-) strand of chromosome 3 (fragment 13Ainv).Blast results of soft-clipped read sequences at chromosome 3 were not informative due to the presence of repeated sequences.
Figure S4 IGV exploration of the fragments from chromosomes 4, 13, and 15 that compose the derivative chromosome 3. Dashed vertical lines indicate breakpoints of each fragment.Panels A-D show the mate regions of the four breakpoints junction (see Table1 and Figure 2 in the main text) identified by OGM (see TableS3).Discordant reads that map to the left (A, B) or right (C, D) region of the breakpoint have mate pairs that map to the positive (+) or negative (-) strand at a second chromosome region.Genomic coordinates of soft-clipped reads are highlighted within boxes.Breakpoint junction sequences, of which two characterized by microhomology of 3-5 bps (A, C) and one by blunt ends (B), are indicated.

Figure
Figure S8 Structural domains and mutation mapping of ROBO2.A schematic representation of the domain structure of ROBO2 is shown.Domain information was retrieved from UniProt (https://www.uniprot.org/)and refers to the Q9HCK4 entry.The location of intrinsically disordered regions is also shown.Missense mutations associated with VUR (black) or CAKUT (congenital anomalies of the kidney and urinary tract, blue) were derived from ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ ) and are mapped onto the domain structure of the protein.The extension of the deletion is reported.

Figure S9
Figure S9 Schematic illustration of the Topologically Associated Domain (TAD) structure encompassing the fragments (indicated with a horizontal gray and red bars) forming the rearrangement (see TableS3), as created by the 3D Genome Browser (http://promoter.bx.psu.edu/hi-c/).The GM12878 Hi-C maps (Ro et al 2014; left) and H1-ESC(Dixon et al. 2015, right)  are shown for each schematic view of TADs.The corresponding region from the UCSC Genome Browser was aligned underneath the heat map.

Table S2 :
Primers used for Sanger sequencing confirmation of breakpoints junctions (Bjct)

Table S3 :
Informative SNPs from the 180K CGH+SNP array platform (G4890A, Agilent Technologies) showing the paternal origin of the 3p12.3deletion

Table S4 :
Chromosome fragments participating in the complex chromosome rearrangement (CCR)